Compositions of citrulline

ABSTRACT

The present disclosure relates generally to supplement compositions containing citrulline and taurine at a weight ratio of 4.56:1 to 2.57:1 which have been demonstrated to have a pleasant flavor and can enhance the exercise endurance in individuals in a synergistic manner.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 16/374,625, filed Apr. 3, 2019, which is acontinuation application of International Application PCT/CN2019/078623,filed Mar. 19, 2019, the contents of each of which are incorporated byreference in their entirety into the present disclosure.

BACKGROUND

L-citrulline, or simply citrulline, is an α-amino acid, with its namederived from citrullus, the Latin word for watermelon. Citrulline is akey intermediate in the urea cycle, the pathway by which mammals excreteammonia by converting it into urea. Citrulline is also produced as abyproduct of the enzymatic production of nitric oxide from the aminoacid arginine, catalyzed by nitric oxide synthase.

Citrulline boosts nitric oxide (NO) production in the body. Nitric oxidehelps arteries relax and work better, which improves blood flowthroughout the body. NO is an integral part of the inflammatory phase,functioning as a regulatory mechanism to mediate epithelialization,angiogenesis, and collagen deposition crucial to the proliferativephase. NO-induced vasodilatation acted as a host-protective agent bykilling pathogens and by increasing blood flow to wounds. The supplementof citrulline is used to lower blood pressure in people withprehypertension. Citrulline supplements may also ease symptoms ofmild-to-moderate erectile dysfunction (ED) and help to address bloodvessel problems such as slow wound healing due to diabetes.

Bodybuilders use citrulline malate, a formulation of L-citrulline withmalic acid added, as a supplement. It is hypothesized that citrullinemalate can increase NO and growth hormone synthesis, and can thereforepromote improvements in athletic performance and physical energy levels.Such performance enhancement effects of citrulline, however, lackclinical validation.

SUMMARY

The present disclosure, in certain embodiments, provides solidcompositions and drinks that contain citrulline and taurine. As theaccompanying experimental examples show, citrulline was able to enhancethe physical performance of mice, and the addition of taurinesignificantly and synergistically boosted the enhancement even further.By contrast, gamma-aminobutyric acid (GABA), which would have beenthought to be more likely to synergize with citrulline, did not evenproduce additive effects.

Also, even though taurine has a strong unpleasant astringent bittertaste in solutions, when it was added to citrulline, it unexpectedlyimproved the tastes of citrulline which on its own only has a mildunnatural taste in solutions. When more taurine was added (e.g., whenthere was more taurine than citrulline in the solution) its astringentbitter taste overshadows the milder taste of citrulline. Taurine'sability to improve the taste of citrulline in solutions, at suitableweight ratios is another surprising discovery of the present disclosure.

In one embodiment, therefore, the present disclosure provides acomposition comprising citrulline and taurine. In some embodiments, thecitrulline and the taurine have a weight ratio of 5:1 to 1:5, or 5:1 to1:1, or 4.56:1 to 2.57:1. In some embodiments, citrulline and taurinetogether constitute at least 20%, 30%, 40%, 50%, 55%, 60%, 70%, 80% or90% w/w of the solid composition or solid portion of the composition. Insome embodiments, the composition is a solid composition.

In some embodiment, the solid composition does not include more than30%, or 20% w/w of the combination of any other amino acid, vitamins,peptides, proteins, herb extracts, fatty acids, fibers, probiotics,glucosamine, chondroitin, and CoQ10. In some embodiments, the solidcomposition does not include more than 10% or 5% w/w malate. In someembodiments, the solid composition does not include more than 10% or 5%w/w arginine. In some embodiments, the solid composition does notinclude more than 30% of 20% w/w of the combination of arginine, malate,niacin, agmatine, alanine, gamma-aminobutyric acid (GABA), creatine, anddextrin.

In some embodiments, the solid composition does not include more than10% or 5% w/w of any other nutritional supplement or activity-enhancingsupplement. In some embodiments, the solid composition does not includemore than 1% w/w of any other nutritional supplement oractivity-enhancing supplement.

In some embodiments, the solid composition further comprises caffeine.In some embodiments, the solid composition further comprises a mineral.In some embodiments, the mineral is selected from calcium, potassium,magnesium, iron, zinc, copper, chromium, selenium, molybdenum, cobalt,nickel, vanadium, tin, strontium, or rubidium.

In some embodiments, the citrulline and the taurine have a weight ratioof 4:1 to 2.85:1. In some embodiments, the citrulline and the taurinehave a weight ratio of 3.55:1 to 3:1. In some embodiments, thecitrulline and the taurine have a weight ratio of 3.35:1 to 3.08:1. Insome embodiments, the citrulline and the taurine have a weight ratio ofabout 3.17:1.

In some embodiments, the citrulline and the taurine together constituteat least 60% w/w of the solid composition. In some embodiments, thecitrulline and the taurine together constitute at least 75% w/w of thesolid composition.

In some embodiments, the solid composition further comprises asweetener, a stabilizer, a binder, a coloring agent, and/or ananticaking agent.

In some embodiments, provided is a drink obtainable by dissolving thesolid composition of the present disclosure in water or an aqueoussolution.

Also provided, in some embodiments, are methods of using thecompositions for improving the athletic performance of a mammaliansubject. Also provided, in some embodiments, are methods of using thecompositions for or treating or preventing prehypertension,hypertension, or erectile dysfunction (ED).

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings described herein are for illustration purposes only. Thedrawings are not intended to limit the scope of the present disclosure.

FIG. 1 shows a lack of synergistic effect between citrulline andgamma-aminobutyric acid (GABA) on exercise time to exhaustion. Mice weregiven either normal saline (control), citrulline (200 mg/kg bodyweight), or GABA (150 mg/kg body weight), or citrulline/taurine mixture(200 mg/kg+150 mg/kg) 1 hour prior to the test. The mice swam withweights attached to their tails, corresponding to ⅛ of their bodyweight, and the swimming time to exhaustion were recorded. Valuesrepresent means±SE.

FIG. 2 shows the synergistic effect of citrulline and taurine onexercise time to exhaustion. Mice were given either normal saline(control), citrulline (200 mg/kg body weight), or taurine (200 mg/kgbody weight), or citrulline/taurine mixture (200 mg/kg+200 mg/kg) 1 hourprior to the test. The mice swam with weights attached to their tails,corresponding to ⅛ of their body weight, and the swimming time toexhaustion were recorded. Values represent means±SE.

FIG. 3 shows the synergistic effect of citrulline and taurine, on lowerdoses, on exercise time to exhaustion. Mice were given either normalsaline (control), or citrulline (75 mg/kg body weight), or taurine (25mg/kg body weight), or citrulline taurine mixture (75 mg/kg+25 mg/kg) 1hour prior to the test. The mice swam with weights attached to theirtails, corresponding to ⅛ of their body weight, and the swimming time toexhaustion were recorded. Values represent means±SE.

FIG. 4 charts the results of a taste survey of various drinks havingdifferent ratios of citrulline and taurine.

FIG. 5 charts the results of a second taste survey of various drinkshaving different ratios of citrulline and taurine.

Reference is now made in detail to certain embodiments of the presentdisclosure. While certain embodiments of the present disclosure aredescribed, it will be understood that it is not intended to limit theembodiments of the present disclosure to the disclosed embodiments. Tothe contrary, reference to embodiments of the present disclosure isintended to cover alternatives, modifications, and equivalents as may beincluded within the spirit and scope of the embodiments of the presentdisclosure as defined by the appended claims.

DETAILED DESCRIPTION

The following description sets forth exemplary embodiments of thepresent technology. It should be recognized, however, that suchdescription is not intended as a limitation on the scope of the presentdisclosure but is instead provided as a description of exemplaryembodiments.

The potential ability of citrulline to boost endurance performance wasassessed with a loaded swimming test in mice. Mice administered with 200mg/kg citrulline exhibited an increase of about 26% in endurance time onaverage (from about 194.7 seconds in control to about 245.4 seconds).Another commonly used athletic supplement, gamma-aminobutyric acid(GABA), was able to increase the endurance time too, but at a moremodest rate of 11.4%.

The addition of GABA to citrulline resulted in a slightly higher effectthan citrulline alone (31% vs. 26%). This slight increase suggests nobenefit of the combination given the small benefit based on the almostdoubled dosage (350 mg/kg vs. 200 mg/kg).

Surprisingly, when citrulline (200 mg/kg) and taurine (200 mg/kg) wereused together, the combinatory effect almost tripled each agent alone(63.1% in combination vs. 15.6% for citrulline and 23.6% for taurinealone). This is clear indication of synergism, which is surprising andunexpected on its own, and more so because of the lack of synergismbetween citrulline and GABA. Moreover, the magnitude of the synergism(63.1% observed vs. a 35.5% theoretical synergism threshold) is evenmore surprising.

At a greatly reduced dose of 25 mg/kg, the effect of taurine alone wasless pronounced (181 seconds as compared to 168.3 seconds in controls, amere 7.5% increase). The effect of citrulline, at 75 mg/kg, was alsoless significant (207.7 seconds). Here, the increases by citrulline andtaurine were 23.4% and 7.5%, respectively. The combination treatment,however, resulted in a 53.7% increase, which further underscores thesynergism between these two amino acids.

Taurine, however, has an unpleasant astringent bitter taste in asolution. The addition of taurine to citrulline, therefore, may not be awelcome change, even though citrulline itself has a sense of unnaturalsweetness. In another surprising discovery of the present disclosure, anaqueous solution of citrulline and taurine in a weight ratio of about2.5:1 to about 4.5:1, in particular at about 3.17:1, has the best taste.More strikingly, the taste of the mixture at the suitable ratio isbetter than each of citrulline and taurine alone. In other words,citrulline and taurine can neutralize each other's unpleasant flavor,and this was entirely unexpected.

In accordance within one embodiment of the present disclosure,therefore, provided is a solid composition comprising citrulline andtaurine. In some embodiments, a solution that contains the solidcomposition dissolved in the solution is also provided. Methods ofpreparing the solid composition or the solution are also provided, invarious embodiments.

In some embodiments, the citrulline and the taurine have a weight ratiofrom about 5:1 to about 1:5. In some embodiments, the weight ratiobetween citrulline and the taurine is from about 5:1, 4.5:1, 4:1, 3.5:1,3:1, 2.5:1, 2:1 1.5:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1,0.3:1, 0.25:1, or 0.2:1 to about 1:5, 1:4.5, 1:4, 1:3.5, 1:3, 1:2.5,1:2, 1:1.5, 1:1, 1:0.9, 1:0.8, 1:07, 1:0.6, 1:0.5, 1:0.4, 1:0.3 or 1:02.

In some embodiments, the citrulline and the taurine have a weight ratioof about 4.56:1 to about 2.57:1. In some embodiments, the citrulline andthe taurine have a weight ratio of about 4:1 to about 2.85:1. In someembodiments, the citrulline and the taurine have a weight ratio of about3.55:1 to about 3:1. In some embodiments, the citrulline and the taurinehave a weight ratio of about 3.35:1 to about 3.08. In some embodiments,the citrulline and the taurine have a weight ratio of about 3.17.

In some embodiments, the citrulline and the taurine have the weightpercentages or weight ratios as shown in Table 1.

TABLE 1 Preferred Taurine % or Citrulline/Taurine Ratios Weight % oftaurine Weight Ratio in total (citrulline/taurine) low high low highPreferred 18 28 4.56 2.57 More Preferred 20 26 4.00 2.85 Even MorePreferred 22 25 3.55 3.00 Most Preferred 23 24.5 3.35 3.08

In some embodiments, the citrulline and the taurine in combinationconstitute the substantial portion of the solid composition. In someembodiments, the citrulline and the taurine in combination constitute atleast about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,or 99% of the solid composition.

In some embodiments, the composition does not include a significantportion of other commonly used nutritional or parametrical activeagents. Examples of nutritional or parametrical active agents includeother amino acids, vitamins, peptides, proteins, herb extracts, fattyacids, fibers, probiotics, glucosamine, chondroitin, and CoQ10. In someembodiments, the composition contains less than about 40%, 35%, 30%,25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% (w/w) of othernutritional or parametrical active agents. In some embodiments, thecomposition contains less than about 40%, 35%, 30%, 25%, 20%, 15%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% (w/w) of other amino acids.

In some embodiments, the composition contains less than about 40%, 35%,30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% (w/w)malate. In some embodiments, the composition contains less than about40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%(w/w) arginine. In some embodiments, the composition contains less thanabout 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%or 1% (w/w) the combination of arginine, malate, niacin, agmatine,alanine and dextrin. In some embodiments, the composition contains lessthan about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2% or 1% (w/w) creatine.

In some embodiments, the composition contains less than about 40%, 35%,30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% (w/w)other activity/performance-enhancing supplements.

The composition can further include one or more other excipients oringredients. Non-limiting examples of excipients include diluents,disintegrants, binders, lubricants, sweeteners, flavoring agents andanti-cake agents.

Diluents act as fillers in nutraceutical/pharmaceutical tablets toincrease weight and improve content uniformity. Examples include,without limitation, microcrystalline cellulose, powdered cellulose,pregelatinized starch, starch, lactitol, mannitol, sorbitol,maltodextrin and combinations thereof.

Disintegrants are agents added to solid formulations (e.g., tablets) topromote the breakup of the tablet (or capsule “slugs”) into smallerfragments in an aqueous environment thereby increasing the availablesurface area and promoting a more rapid release of the drug substance.Examples include, without limitation, croscarmellose sodium,crospovidone, starch, sodium carboxymethyl starch, low-substitutedhydroxypropyl cellulose and combinations thereof.

Binders are formulated to act as an adhesive to bind together powders,granules and other dry ingredients to impart to the product thenecessary mechanical strength. Examples include, without limitation,distilled water, ethanol, hypromellose, sodium carboxymethylcellulose,povidone, ethyl cellulose and combinations thereof.

Lubricants are agents added to tablet and capsule formulations toimprove the powder processing properties of the formulation. Lubricants(glidants) can enhance the powder flow by reducing the inter-particlefriction. Non-limiting examples include sodium stearyl fumarate,magnesium stearate, calcium stearate, stearic acid, glyceryl behenate,and combinations thereof.

Non-limiting examples of sweeteners include xylitol, sorbitol, mannitol,maltitol, lactitol, isomalt, erythritol, aspartame, acesulfamepotassium, alitame, sodium saccharin, three Sucralose, neotame,cyclamate, and combinations thereof.

Non-limiting examples of flavoring agents include orange flavor, orangeflavor, mango flavor, peach flavor, grapefruit flavor, and combinationsthereof.

Anti-caking agents are additives placed in powdered or granulatedmaterials, such as table salt, to prevent the formation of lumps and foreasing packaging, transport, and consumption. Anticaking agents functioneither by adsorbing excess moisture, or by coating particles and makingthem water repellent. Non-limiting examples of anti-caking agentsinclude silica, tricalcium phosphate, microcrystalline cellulose andcombination thereof.

In addition, in effervescent tablet or granules, an agent that producescarbon dioxide Is needed, and their examples include acidic materialssuch as citric acid, tartaric acid, fumaric acid, malic acid, adipicacid, succinic acid, ascorbic acid, maleic acid, and combinationsthereof. Further included in the effervescent agent are alkali metalhydrogencarbonates including sodium hydrogencarbonate, potassiumhydrogencarbonate, and combinations thereof.

In some embodiments, the composition further includes caffeine. In someembodiments, the composition further includes a mineral, such ascalcium, potassium, magnesium, iron, zinc, copper, chromium, selenium,molybdenum, cobalt, nickel, vanadium, tin, strontium, or rubidium.

The solid compositions of the present disclosure can be provided astablets, capsules, powders, granules, or effervescent tablets orgranules. Solutions (e.g., beverages) that include citrulline and thetaurine are also provided.

The solutions can be prepared by dissolving a solid composition of thepresent disclosure in water or an aqueous solution. In some embodiments,the solution includes from about 0.1% (w/w) to about 20% (w/w) of acombination of citrulline and the taurine. In some embodiments, thesolution includes at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%,1.9%, or 2% (w/w) of the combination of citrulline and the taurine. Insome embodiments, the solution includes no more than about 20%, 18%,15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%,2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%,1.2%, 1.1%, or 1% of the combination of citrulline and the taurine.

In some embodiments, the solution has a pH of about 2.0 to about 9.0. Insome embodiments, the solution has a pH of at least about 2.0, 2.5, 3.0,3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7. In some embodiments, the solution hasa pH of not higher than about 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5 or4. In some embodiments, the pH is from about 2.5 to about 3.5. In someembodiments, the pH is from about 3 to 4. In some embodiments, the pH isfrom about 3.5 to 4.5. In some embodiments, the pH is from about 4 to 5.In some embodiments, the pH is from about 5 to 6. In some embodiments,the pH is from about 6 to 7. In some embodiments, the pH is from about 7to 8. In some embodiments, the pH is from about 8 to 9.

In some embodiments, the citrulline and the taurine in the solution havea weight ratio of about 4.56:1 to about 2.57:1. In some embodiments, thecitrulline and the taurine in the solution have a weight ratio of about4:1 to about 2.85:1. In some embodiments, the citrulline and the taurinein the solution have a weight ratio of about 3.55:1 to about 3:1. Insome embodiments, the citrulline and the taurine in the solution have aweight ratio of about 3.35:1 to about 3.08. In some embodiments, thecitrulline and the taurine in the solution have a weight ratio of about3.17.

Specific examples of solid compositions and solutions disclosed hereinclude, without limitation, those provided in Experimental Example 5.

Methods for preparing and using the aqueous solutions of the presentdisclosure are also provided. In some embodiments, the solutions can beprepared by adding each of the ingredients into a water-based solution.

In various embodiments, the solid compositions and solutions disclosedherein can be used in methods for enhancing stamina, athleticperformance, endurance, and/or reducing muscle soreness, fatigue orcramping in a subject in need thereof. In some embodiments, the solidcompositions and solutions disclosed herein can be used in methods forpreventing or treating hypertension or prehypertension in a subject inneed thereof. In some embodiments, the solid compositions and solutionsdisclosed herein can be used in methods for preventing or treatingerectile dysfunction (ED) in a male subject in need thereof. The method,in some embodiments, entails orally administering to the subject aneffective amount of the solid composition or solution of the presentdisclosure.

In some embodiments, provided is a method or use for improving theathletic performance of a mammalian subject. In some embodiments,provided is a method or use for enhancing the physical endurance orreducing fatigue of a mammalian subject. In some embodiments, providedis a method or use for enhancing the muscular strength of a mammaliansubject.

In some embodiments, provided is a method or use for treating orpreventing erectile dysfunction (ED) in a male mammalian subject. Insome embodiments, wherein the ED is mild or moderate. In someembodiments, provided is a method or use for treating or preventinghypertension or prehypertension in a mammalian subject.

In various embodiments, the method entails administering, in particularorally, to the subject a solid composition or a drink of the disclosure.In some embodiments, about 0.1 g to about 100 g of the combination ofcitrulline and the taurine are administered daily. In some embodiments,the daily dose of the combination of citrulline and the taurine is atleast 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 15 g, 20 g, 30 g, 40 g,or 50 g. In some embodiments, the daily dose of the combination ofcitrulline and the taurine is not greater than 100 g, 90 g, 80 g, 70 g,60 g, 50 g, 40 g, 30 g, 20 g, 15 g, 10 g, 9 g, 8 g, 7 g, 6 g, 5 g, 4 g,3 g, 2 g or 1 g.

In some embodiments, the solid composition or the drink is administeredonce, twice or three times daily. In some embodiments, the solidcomposition or the drink is administered prior to, during, or followingphysical activities, or prior to a sexual activity.

In some embodiments, the administration follows an intense physicalactivity by the subject. In one embodiment, the administration is madebefore an intense physical activity by the subject.

In some embodiments, the effective amount of the solution is about 30ml, 50 ml, 75 ml, 100 ml, 120 ml, 150 ml, 200 ml, 250 ml, 300 ml, 400ml, or 500 ml, without limitation. In some embodiments, the effectiveamount of the solid composition is about 0.1, 0.2, 0.5 g, 1 g, 1.5 g, 2g, 3 g, 4 g, 5 g, 10 g, 15 g or 20 g without limitation.

In some embodiments, the subject experiences muscle soreness, fatigue,or cramping. In some embodiments, the subject, following theadministration, experiences reduced muscle soreness, fatigue orcramping. In some embodiment, the subject desires the flavor of thesolution.

Examples

The following examples are included to demonstrate specific embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques to function well in the practice of the disclosure, and thuscan be considered to constitute specific modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the disclosure.

Example 1. Effects of Citrulline and GABA on Endurance and MuscleStrength

This example tested the effect of citrulline on the endurance and musclestrength of mice in exercise, and explores the potential synergisticeffect of gamma-aminobutyric acid (GABA) to citrulline.

Materials and Methods

Animals. Adult (8-10 weeks) male ICR mice were used in this study andpurchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd. All the mice were provided with a normal diet and water ad libitum.All procedures were approved by institutional guidelines and the AnimalCare and Use Committee (Huazhong University of Science and Technology,Wuhan, China) of the university's animal core facility. The mice weregroup-housed under a 12 h light-dark cycle (lights on at 8:00 a.m.), atconsistent humidity (50±5%), and ambient temperature (22˜24° C.).

The animals were bred with three mice per cage in the laboratory, andwere acclimatized for 1 week before the experiments. The mice weregrouped randomly for each experiment. The mice in each group were giventhe corresponding dose amino acid and the control group was treated withthe equal volume of normal saline (NS) through intragastricadministration. Loaded swimming experiments were performed to assess theacute effect of the amino acids on endurance one hour after thetreatment

The NS injection were purchased from Beijing Pharmaceutical Group Co.,Ltd. The amino acid powders were dissolved in NS and then wereintragastric administration to mice by lavage needle (Beijing VitalRiver Laboratory Animal Technology Co., Ltd.).

Loaded Swimming Test

Acute effects on endurance performance were assessed by loaded swimmingtest one hour after the amino acid treatment. The swimming exercise wascarried out in a tank (diameter 120 cm) filled with fresh water to 30-cmdepth at 24±2° C. A tin wire (⅛ of body weight) was attached on the tailof the mouse. Each mouse was considered to have reached exhaustion whenit failed to rise its face to the surface of the water for inhale within5 s period. The length of time each mouse kept swimming beforeexhaustion (exercise time to exhaustion) was recorded. At the end of thesession, the mice were removed from the water, dried with paper towels,and placed back in their home cages.

Results

Sixty mice were randomly divided into four groups (n=15 per group). Eachof the four group were treated with NS, citrulline (200 mg/kg bodyweight), GABA (150 mg/kg body weight), and citrulline/GABA mixture (200mg/kg+150 mg/kg) respectively. The results were summarized below (Table2 and FIG. 1).

TABLE 2 Effects of citrulline and citrulline/GABA combination onexercise time to exhaustion Control Citrulline GABA Citrulline + GABAAverage Time (s) 194.7 245.4 216.8 255 SE 14.67 28.01 37.88 23.03Increase — 26% 11.4% 31%

Citrulline increased the endurance time, as compared to vehicle control,by about 26%. GABA alone also increased the endurance, though at aslightly lower rate, at about 11.4%. The combination of citrulline andGABA resulted in a 31% increase.

A commonly accepted way to estimate the combinatory effect, withoutsynergism, is Ec=Ea+Eb−Ea*Eb, where Ea, Eb and Ec represent the effectsof agents a and b and their combination, respectively. Here, theestimated combinatory effect without synergism would be a 34.4% increase(vs. a sum of both values being 37.4%). In FIG. 1, the predictedsynergism threshold (261.7 seconds) is indicated as a horizontal dottedline. The observed combinatory effect was an endurance enhancement of31% (255 seconds), suggesting that there was no synergism betweencitrulline and GABA. Also, given the modest added effect of GABA (5%over citrulline's 26%) from the 150 mg/kg dose, GABA does not appear tobe promising additive to citrulline.

This result was disappointing as GABA was suggested to have the abilityto enhance muscle building. Further, according to Shyamaladevi et al.,Brain Research Bulletin 57(2):231-6 (2002), for instance, nitric oxide(NO) increases the blood-brain barrier permeability for GABA. Meanwhile,citrulline boosts NO production in the body. Therefore, when citrullineis supplied to the body, it would have been expected to raise theavailability of GABA in the brain. The interaction between the twoagents, therefore, would have been expected to exhibit synergy. Suchsynergy, however, was not observed.

Example 2. Unexpected Synergy Between Citrulline and Taurine onEndurance and Muscle Strength

This example continued to look for agents that could significantlyfurther increase citrulline's activity in enhancing the endurance andmuscle strength of mice in exercise. Taurine was surprisingly found tohave such synergism with citrulline.

The methods used here are the same as in Example 1, but GABA (150 mg/kg)was replaced by taurine (200 mg/kg). The results were summarized below(Table 3 and FIG. 2).

TABLE 3 Effect of citrulline taurine combination on exercise time toexhaustion Control Citrulline Taurine Citrulline + Taurine Average Time(s) 168.3 194.6 208 274.5 SE 12.58 17.02 22.20 20.33 Increase — 15.6%23.6% 63.1%

Citrulline and taurine increased the endurance time by 15.6% and 23.6%,respectively. As the synergism estimation methodology explained inExample 1, the estimated combinatory effect without synergism would be a35.5% increase (vs. a sum of both values being 39.2%). In FIG. 2, thepredicted synergism threshold (228.1 seconds) is indicated as ahorizontal dotted line. The observed combinatory effect, however, was anendurance enhancement of 63.1% (274.5 seconds), clearly indicating astrong synergy between these two amino acids.

Example 3. Synergy Between Lower Doses of Citrulline and Taurine onEndurance and Muscle Strength

This example tested whether the synergism between citrulline and taurineis still present in lower doses, and confirmed the synergism.

The methods used here are the same as in Example 1. Sixty mice wererandomly divided into four groups (n=15 per group). Each of the fourgroup were treated with NS, citrulline (75 mg/kg body weight), taurine(25 mg/kg body weight), and citrulline taurine mixture (75 mg/kg+25mg/kg) respectively. The results were summarized below (Table 4 and FIG.3).

TABLE 4 Effect of lower doses of citrulline taurine combination onexercise time to exhaustion. Control Citrulline Taurine Citrulline +Taurine Average Time (s) 168.3 207.7 181 258.7 SE 18.53 16.67 16.5622.05 Increase — 23.4% 7.5% 53.7%

Here, the lower doses of citrulline and taurine increased the endurancetime by 23.4% and 7.5%, respectively. The estimated combinatory effectwithout synergism would be a 29.1% increase (vs. a sum of both valuesbeing 30.9%). In FIG. 3, the predicted synergism threshold (217.4seconds) is indicated as a horizontal dotted line. The observedcombinatory effect, however, was an endurance enhancement of 53.7%(258.7 seconds), again a showing of strong synergy between these twoamino acids even at the lowered doses.

Further, Examples 2 and 3 demonstrate that the synergism betweencitrulline and taurine exist at different dose levels and at differentratios (e.g., 1:1 and 3:1).

Example 4. Taste Surveys

Citrulline solution does not have a strong taste, but has a sense ofunnatural sweetness that is distinguishable. The taurine solution,however, clearly has an astringent bitter taste, even at a fairlydiluted concentration. The mixture of citrulline and taurine, therefore,presents a challenge in terms of flavoring, as compared to citrullinealone. This example explored options to minimize the unpleasant tastewith different ratios of these two amino acids.

Taste Survey No. 1 Method

Six different citrulline-taurine mixtures were prepared withcitrulline/taurine ratios ranging from 8/0, 7/1, 4/1, 6/2, 5/3, to 4/4.Testing samples were prepared by dissolving 6 grams of each mixture in250 ml drinking water separately. Thirty volunteers, 12 females and 18males, aged from 22-55 participated in the test. The volunteers wereasked to taste each sample without knowing the composition of thesamples. Each volunteer sorted the samples from 1 to 6, with 1 being themost liked and 5 being the least liked sample.

Results

The survey results are shown in Table 5.

TABLE 5 Survey results Citrulline:Taurine Ratio Individual 8:0 7:1 4:16:2 5:3 4:4 P01 6 5 4 3 1 2 P02 4 3 2 1 6 5 P03 4 3 1 2 5 6 P04 6 3 2 14 5 P05 4 3 1 2 5 6 P06 5 2 3 1 4 6 P07 6 4 3 1 2 5 P08 5 6 4 2 1 3 P094 3 2 1 5 6 P10 5 3 2 1 4 6 P11 3 4 5 1 2 6 P12 6 4 2 1 3 5 P13 4 3 2 16 5 P14 2 3 4 1 5 6 P15 4 2 1 3 6 5 P16 6 5 3 1 2 4 P17 3 4 2 1 5 6 P186 4 5 3 1 2 P19 4 2 6 5 3 1 P20 1 3 5 6 2 4 P21 3 4 2 5 1 6 P22 6 3 1 24 5 P23 4 3 1 2 6 5 P24 2 4 3 1 5 6 P25 4 1 3 2 6 5 P26 5 4 1 2 3 6 P274 3 1 2 5 6 P28 4 1 3 5 2 6 P29 4 3 1 2 5 6 Total 124 95 75 61 109 145

The results are plotted in FIG. 4, which indicates that when citrullineand taurine were mixed at about a 3:1 weight ratio, the taste was themost favorable. What was entirely unexpected and even striking is thatthe mixtures had more favorable tastes than each of its ingredientsalone. For instance, citrulline alone (i.e., 8:0 citrulline to taurine)had a taste survey score of 124. When taurine was added, the tastesurvey score improved to 95 (7:1), 75 (4:1) and 61 (6:2) and thendeteriorated when more taurine was added. In other words, addition ofthe more unfavorable taurine was able to even reduce the lessunfavorable taste of citrulline.

To further investigate the tastes of citrulline taurine combinations,nine different citrulline-taurine mixtures were prepared with taurinecontent in the mixture of 20%, 22%, 24%, 25%, 26%, 28%, 30%, 32%, and34%. Testing samples were prepared by dissolving 6 grams of each mixturein 250 ml drinking water separately. Twenty volunteers, 11 females and 9males, aged from 22-55 participated in the test. The volunteers wereasked to taste each sample without knowing the composition of thesamples. Each volunteer sorted the samples from 1 to 9, with 1 being themost liked and 5 being the least liked sample. The survey results areprovided in Table 6 below.

TABLE 6 Survey results Indi- Taurine content in the mixture vidual 20%22% 24% 25% 26% 28% 30% 32% 34% Q01 8 2 1 6 9 7 5 4 3 Q02 1 2 3 5 9 4 68 7 Q03 3 2 1 4 6 7 8 9 5 Q04 9 8 1 7 2 6 4 3 5 Q05 2 3 1 4 5 7 6 9 8Q06 4 3 5 1 2 6 7 9 8 Q07 4 1 2 3 5 7 6 9 8 Q08 2 1 4 5 3 6 7 8 9 Q09 54 2 3 1 6 7 8 9 Q10 5 2 1 3 4 7 6 8 9 Q11 3 1 2 4 5 6 7 9 8 Q12 7 6 5 29 4 3 1 8 Q13 3 1 2 4 5 6 7 9 8 Q14 7 6 5 4 3 2 1 8 9 Q15 3 1 2 6 5 4 87 9 Q16 4 3 2 1 5 6 7 9 8 Q17 5 2 3 1 4 7 6 8 9 Q18 5 4 2 3 1 6 7 8 9Q19 3 2 4 1 8 6 9 7 5 Total 83 54 48 67 91 110 117 141 144

As the composition difference between samples gets smaller, the curvebecame shallower. However, the result indicates that samples withtaurine content between 20% and 28% were best “liked”, with 24% beingthe winner (FIG. 5).

Example 5. Production Samples

This example lists a number of production samples in different forms(solid or in solution).

Production Sample 1 (Powder):

Ingredient Amount Unit Citrulline 76 parts Taurine 24 parts Maltodextrin1.5 parts Microcrystalline cellulose 1 parts Food flavor 1 parts

Production sample 1 is prepared as follows. The ingredients are weighedaccording to the formula, and are mixed together in a uniform manner.Alternatively, they are added into water is then spray dried.

Production Sample 2 (Oral Solution):

Ingredient Amount Unit Citrulline 1.52 % Taurine 0.48 % Creatinemonohydrate 0.15 % Maltodextrin 0.1 % Concentrated lemon juice 0.005 %Sodium cyclamate 0.0034 % Acesulfame 0.00255 % Sodium bicarbonate 0.001% Aspartame 0.00085 % Orange flavor 0.0005 % Water q.s. to 100 mg %

Production sample 2 is prepared as follows. The ingredients are weighedaccording to the formula, stirred and dissolved uniformly to obtain amixed liquid. The mixture is filtered and then bottled. Sterilization iscarried out to obtain a finished product.

Production Sample 3 (Tablets or Granules):

Ingredient Amount Unit Citrulline 80 parts Taurine 20 parts Filler 25parts Disintegrant 2.5 parts Lubricant 2.5 parts Binder 2.5 partsSweetener 0.1 parts

Production sample 3 is prepared as follows. The ingredients are weighedaccording to the formula and are mixed with water in a wet granulator.The particles are then dried in an oven to obtain granules. The granulescan be pressed to prepare tablets.

Production Sample 4 (Effervescent Granules):

Ingredient Amount Unit Citrulline 66 parts Taurine 33 parts Filler 25parts Citric acid 25 parts Sodium bicarbonate 25 parts

Production sample 4 is prepared as follows. The ingredients are weighedaccording to the formula and are mixed with water in a wet granulator.The particles are then dried in an oven to obtain effervescent granules.

Production Sample 5 (Effervescent Tablets):

Ingredient Amount Unit Citrulline 75 parts Taurine 25 parts Filler 25parts Disintegrant 5 parts Lubricant 5 parts Sweetener 2.5 parts Citricacid 25 parts Sodium bicarbonate 25 parts

Production sample 5 is prepared as follows. The ingredients (exceptcitric acid and sodium bicarbonate) are weighed according to the formulaand are mixed with water in a wet granulator. The particles are thendried in an oven. Citric acid and sodium bicarbonate are then added tothe particles and pressed to prepare effervescent tablets.

Production Sample 6 (Capsules):

Ingredient Amount Unit Citrulline 76 parts Taurine 24 parts Filler 25parts Disintegrant 5 parts Lubricant 5 parts Binder 5 parts

Production sample 6 is prepared as follows. The ingredients (exceptcitric acid and sodium bicarbonate) are weighed according to the formulaand are mixed with water in a wet granulator. The particles are thendried in an oven and filled into capsules in a filling machine.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement and variation of the inventionsembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications, improvements and variations areconsidered to be within the scope of this invention. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

It is to be understood that while the disclosure has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of thedisclosure. Other aspects, advantages and modifications within the scopeof the disclosure will be apparent to those skilled in the art to whichthe disclosure pertains.

1. A solid composition comprising citrulline and taurine, wherein thecitrulline and the taurine have a weight ratio of 5:1 to 1:5 andtogether constitute at least 55% w/w of the solid composition.
 2. Thesolid composition of claim 1, wherein the citrulline and the taurinehave a weight ratio of 5:1 to 1:1.
 3. The solid composition of claim 2,which does not include more than 20% w/w of the combination of any otheramino acid, vitamins, peptides, proteins, herb extracts, fatty acids,fibers, probiotics, glucosamine, chondroitin, and CoQ10.
 4. The solidcomposition of claim 3, wherein the citrulline and the taurine have aweight ratio of 4:1 to 2.85:1.
 5. The solid composition of claim 3,wherein the citrulline and the taurine have a weight ratio of 3.55:1 to3:1.
 6. The solid composition of claim 3, wherein the citrulline and thetaurine have a weight ratio of 3.35:1 to 3.08:1.
 7. The solidcomposition of claim 3, wherein the citrulline and the taurine togetherconstitute at least 60% w/w of the solid composition.
 8. The solidcomposition of claim 3, wherein the citrulline and the taurine togetherconstitute at least 75% w/w of the solid composition.
 9. The solidcomposition of claim 1, which is provided in the form of powders,tablets, granules, or capsules.
 10. The solid composition of claim 9,wherein the tablets or granules are effervescent tablets or granules.11. A drink comprising citrulline and taurine, wherein the citrullineand the taurine have a weight ratio of 5:1 to 1:5 and togetherconstitute at least 55% w/w of total solid ingredients dissolved in thedrink.
 12. The drink of claim 11, wherein the citrulline and the taurinehave a weight ratio of 5:1 to 1:1.
 13. The drink of claim 11, whereinthe citrulline and the taurine have a weight ratio of 4:1 to 2.85:1. 14.The drink of claim 11, wherein the citrulline and the taurine have aweight ratio of 3.55:1 to 3:1.
 15. The drink of claim 11, wherein thecitrulline and the taurine have a weight ratio of 3.35:1 to 3.08:1. 16.The drink of claim 12, wherein the citrulline and the taurine togetherconstitute at least 60% w/w of the total solid ingredients dissolved inthe drink.
 17. The drink of claim 12, wherein the citrulline and thetaurine together constitute at least 75% w/w of the total solidingredients dissolved in the drink.
 18. A method for treatinghypertension or prehypertension in a mammalian subject, comprisingorally administering to the subject the drink of claim
 11. 19. Themethod of claim 18, wherein about 0.1 g to about 100 g of thecombination of citrulline and the taurine are administered daily. 20.The method of claim 18, wherein about 1 g to 10 g of the combination ofcitrulline and the taurine are administered daily.